The alpha subunit of the stimulatory G protein (Gsalpha), and its large variant XLalphas, are derived from GNAS1 through the use of alternative promoters and pre-mRNA splicing. XLalphas, unlike Gsalpha, shows limited tissue distribution, including expression in kidney, and is expressed paternally in all investigated tissues. Despite having distinct amino-terminal domains, the two proteins are identical over a long stretch of carboxyl-terminal amino acids comprising almost all the structural features characterized for Gsalpha. Thus, XLalphas and Gsalpha may have similar functional properties in the cell, and our recently published results are consistent with this hypothesis. Given the importance of Gsalpha in numerous different biological responses, the "Gs-like" activity of XLalphas may also have significant roles in the body. Heterozygous inactivating mutations within Gsalpha-encoding exons of GNASI are associated with multiple phenotypes that show parent-specific inheritance, including pseudohypoparathyroidism (PHP), progressive osseous heteroplasia (POH), and Albright's hereditary osteodystrophy (AHO). After paternal transmission, most of these mutations are predicted to disrupt both Gsalpha and XLalphas, suggesting that pathogenesis of some of these disorders, such as POH, may involve not only the deficiency of Gsalpha but also of XLalphas. Moreover, in disorders that develop after maternal inheritance, such as PHP-Ia, the activity of XLalphas expressed from the intact paternal allele may provide compensatory "Gs-like" signaling in certain cells, thereby contributing to the selectivity of hormone resistance in certain types of PHP. My main objective is thus to further explore the role of the "Gs-like" activity of XLalphas in the molecular and genetic mechanisms underlying these diseases. My first specific aim involves a more detailed in vitro characterization of XLas, and to compare its functional properties with those of Gsalpha, particularly regarding possible receptor selectivity and differential effects of naturally-occurring GNASI mutations. I will also identify the unique XLalphas-specific features that are important for its signaling activity. In addition, I will examine spatial and temporal expression of XLalphas in the kidney to therefore provide further insights into possible involvement of XLas in actions mediated by parathyroid hormone in this tissue. Finally, I will determine whether XLalphas can have "Gs-like" activity in vivo, by generating an animal model with targeted expression of XLalphas in the renal proximal tubule. These studies will be helpful in clarifying the biological roles of XLalphas, and may furthermore improve the current understanding of the GNASl-related disorders.